ABSTRACTPneumocystis pneumonia is a major cause of morbidity and mortality among immunocompromised patients, especially in the context of HIV/AIDS. In the murine model of Pneumocystis pneumonia, CD4(+) T-cells are required for clearance of a primary infection of Pneumocystis, but not the memory recall response. We hypothesized that the memory recall response in the absence of CD4(+) T-cells is mediated by a robust memory humoral response, CD8(+) T-cells, and IgG-mediated phagocytosis by alveolar macrophages. To investigate the role of CD8(+) T-cells and alveolar macrophages in the immune memory response to Pneumocystis, mice previously challenged with Pneumocystis were depleted of CD8(+) T-cells or alveolar macrophages prior to re-infection. Mice depleted of CD4(+) T-cells prior to secondary challenge cleared Pneumocystis infection within 48 h identical to immunocompetent mice during a secondary memory recall response. However, loss of CD8(+) T-cells or macrophages prior to the memory recall response significantly impaired Pneumocystis clearance. Specifically, mice depleted of CD8(+) T-cells or alveolar macrophages had significantly higher fungal burden in the lungs. Furthermore, loss of alveolar macrophages significantly skewed the lung CD8(+) T-cell response toward a terminally differentiated effector memory population and increased the percentage of IFN-γ(+) CD8(+) T-cells. Finally, Pneumocystis-infected animals produced significantly more bone marrow plasma cells and Pneumocystis-specific IgG significantly increased macrophage-mediated killing of Pneumocystis in vitro. These data suggest that secondary immune memory responses to Pneumocystis are mediated, in part, by CD8(+) T-cells, alveolar macrophages, and the production of Pneumocystis-specific IgG.

Mentions:
We then assessed the composition and quantity of various lung CD8+ T-cell subsets. We first examined the levels of naive CD8+ T (Figures 4A,E), central memory CD8+ T (Figures 4B,F), effector memory CD8+ T cells (Figures 4C,G), and terminally differentiated effector memory (TEMRA) CD8+ T cells (Figures 4D,H) in mice depleted of CD4+ T-cells, alveolar macrophages, or CD4+ T-cells and alveolar macrophages prior to a secondary infection at 2 and 4 days post-secondary infection. The gating strategy used to determine the various CD8+ T-cell phenotypes are depicted in Figure 4I. We defined the CD8+ T-cell subsets as naive (CD45+CD3+CD4−CD8+CD45RA+CD28+), central memory (CD45+CD3+CD4−CD8+CD45RA−CD197+), effector memory (CD45+CD3+CD4−CD8+CD45RA−CD197−), and TEMRA (CD45+CD3+CD4−CD8+CD45RA+CD28−). Interestingly, loss of CD4+ T-cells did not significantly affect the percentage of any of the CD8+ T-cell subsets, suggesting that CD8+ T-cell immune memory remains active despite loss of CD4+ T-cells. However, mice depleted of alveolar macrophages prior to a secondary infection exhibited a significant decrease in the percentage of effector memory CD8+ T-cells, as well as a significant increase in the percentage of TEMRA CD8+ T cells at both 2 and 4 DPI. We also found that mice depleted of macrophages and CD4+ T-cells prior to a secondary infection exhibited a significant decrease in the amount of naive CD8+ T-cells at 2 DPI, and this trend remained at 4 DPI. The percentage of central memory CD8+ T cells in the lung 2 DPI in immune-intact animals did not significantly differ from CD4-depleted animals or macrophage-depleted mice. However, by 4 DPI, the percentage of central memory CD8+ T cells significantly decreased in macrophage-depleted mice. These results suggest that loss of CD4+ T-cells does not affect the immune memory response of CD8+ T-cells, but that loss of macrophages or loss of both macrophages and CD4+ T-cells drives the CD8+ T-cell response toward a terminally differentiated effector memory phenotype.

Mentions:
We then assessed the composition and quantity of various lung CD8+ T-cell subsets. We first examined the levels of naive CD8+ T (Figures 4A,E), central memory CD8+ T (Figures 4B,F), effector memory CD8+ T cells (Figures 4C,G), and terminally differentiated effector memory (TEMRA) CD8+ T cells (Figures 4D,H) in mice depleted of CD4+ T-cells, alveolar macrophages, or CD4+ T-cells and alveolar macrophages prior to a secondary infection at 2 and 4 days post-secondary infection. The gating strategy used to determine the various CD8+ T-cell phenotypes are depicted in Figure 4I. We defined the CD8+ T-cell subsets as naive (CD45+CD3+CD4−CD8+CD45RA+CD28+), central memory (CD45+CD3+CD4−CD8+CD45RA−CD197+), effector memory (CD45+CD3+CD4−CD8+CD45RA−CD197−), and TEMRA (CD45+CD3+CD4−CD8+CD45RA+CD28−). Interestingly, loss of CD4+ T-cells did not significantly affect the percentage of any of the CD8+ T-cell subsets, suggesting that CD8+ T-cell immune memory remains active despite loss of CD4+ T-cells. However, mice depleted of alveolar macrophages prior to a secondary infection exhibited a significant decrease in the percentage of effector memory CD8+ T-cells, as well as a significant increase in the percentage of TEMRA CD8+ T cells at both 2 and 4 DPI. We also found that mice depleted of macrophages and CD4+ T-cells prior to a secondary infection exhibited a significant decrease in the amount of naive CD8+ T-cells at 2 DPI, and this trend remained at 4 DPI. The percentage of central memory CD8+ T cells in the lung 2 DPI in immune-intact animals did not significantly differ from CD4-depleted animals or macrophage-depleted mice. However, by 4 DPI, the percentage of central memory CD8+ T cells significantly decreased in macrophage-depleted mice. These results suggest that loss of CD4+ T-cells does not affect the immune memory response of CD8+ T-cells, but that loss of macrophages or loss of both macrophages and CD4+ T-cells drives the CD8+ T-cell response toward a terminally differentiated effector memory phenotype.

ABSTRACTPneumocystis pneumonia is a major cause of morbidity and mortality among immunocompromised patients, especially in the context of HIV/AIDS. In the murine model of Pneumocystis pneumonia, CD4(+) T-cells are required for clearance of a primary infection of Pneumocystis, but not the memory recall response. We hypothesized that the memory recall response in the absence of CD4(+) T-cells is mediated by a robust memory humoral response, CD8(+) T-cells, and IgG-mediated phagocytosis by alveolar macrophages. To investigate the role of CD8(+) T-cells and alveolar macrophages in the immune memory response to Pneumocystis, mice previously challenged with Pneumocystis were depleted of CD8(+) T-cells or alveolar macrophages prior to re-infection. Mice depleted of CD4(+) T-cells prior to secondary challenge cleared Pneumocystis infection within 48 h identical to immunocompetent mice during a secondary memory recall response. However, loss of CD8(+) T-cells or macrophages prior to the memory recall response significantly impaired Pneumocystis clearance. Specifically, mice depleted of CD8(+) T-cells or alveolar macrophages had significantly higher fungal burden in the lungs. Furthermore, loss of alveolar macrophages significantly skewed the lung CD8(+) T-cell response toward a terminally differentiated effector memory population and increased the percentage of IFN-γ(+) CD8(+) T-cells. Finally, Pneumocystis-infected animals produced significantly more bone marrow plasma cells and Pneumocystis-specific IgG significantly increased macrophage-mediated killing of Pneumocystis in vitro. These data suggest that secondary immune memory responses to Pneumocystis are mediated, in part, by CD8(+) T-cells, alveolar macrophages, and the production of Pneumocystis-specific IgG.